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. 2012 Sep;167(1):26-36.
doi: 10.1111/j.1476-5381.2012.01934.x.

Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

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Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

Lei Yu et al. Br J Pharmacol. 2012 Sep.

Abstract

Background and purpose: Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K(+) channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K(ATP) channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K(ATP) channels are also sensitive to rosiglitazone and what their sensitivities are.

Experimental approach: K(IR) 6.1/SUR2B, K(IR) 6.2/SUR1, K(IR) 6.2/SUR2A, K(IR) 6.2/SUR2B and K(IR) 6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques.

Key results: Rosiglitazone inhibited all isoforms of K(ATP) channels in excised patches and in the whole-cell configuration. Its IC(50) was 10 µmol·L(-1) for the K(IR) 6.1/SUR2B channel and ∼45 µmol·L(-1) for K(IR) 6.2/SURx channels. Rosiglitazone also inhibited K(IR) 6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC(50) = 45 µmol·L(-1) ) almost identical to that for K(IR) 6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K(IR) 1.1, K(IR) 2.1 and K(IR) 4.1 channels, suggesting that the channel inhibitory effect is selective for K(IR) 6.x channels.

Conclusions and implications: These results suggest a novel K(ATP) channel inhibitor that acts on the pore-forming K(IR) 6.x subunit, affecting the channel gating.

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Figures

Figure 1
Figure 1
Inhibition of KIR6.1/SUR2B channel by rosiglitazone (RSG) in an inside-out patch. (A) An HEK cell was co-transfected with KIR6.1 and SUR2B. The holding potential for the patch was −60 mV. The channels were activated by 10 µM pinacidil (Pin) and then dose-dependently inhibited by rosiglitazone. Washout led to complete recovery. (B) The conductance of KIR6.1/SUR2B channel (35 pS) was not changed after a treatment with 10 µM rosiglitazone (C, 35 pS).
Figure 2
Figure 2
Concentration-dependent inhibition of three KIR6.2 channel isoforms by rosiglitazone (RSG). At baseline, all KIR6.2-containing channels were active without ATP and KATP channel opener. Exposure to rosiglitazone produced dose-dependent inhibition of the KIR6.2/SUR2B (A), KIR6.2/SUR1 (B) and KIR6.2/SUR2A (C). Complete channel inhibition was seen with 1 mM ATP. The channel inhibition was reversible, and the current amplitudes almost returned to baseline levels after washout (WS). Note that 8 superimposed traces are shown in each panel.
Figure 3
Figure 3
The relationship of channel activity with rosiglitazone (RSG) concentration was described using Equation 2. All combinations of KIR6.x and SURx subunits showed clear concentration dependence. The IC50 was 10 µM for KIR6.1/SUR2B (h 1.3, n= 10 patches), 45 µM for KIR6.2/SUR1 (h 1.2, n= 5), 37 µM for KIR6.2/SUR2A (h 1.1, n= 5), 50 µM for KIR6.2/SUR2B (h 1.2, n= 6–7) and 45 µM for KIR6.2ΔC36 (h 1.3, n= 5–8). See the text for the h values.
Figure 4
Figure 4
KIR6.2ΔC36 also showed dose-dependent inhibition by rosiglitazone (RSG) (A) The channel inhibition was reversible with washout (WS). Also, the unitary conductance was not changed with 100 µM rosiglitazone treatment, which was 74 pS with or without RSG (B).
Figure 5
Figure 5
Rosiglitazone (RSG) had no inhibitory effect on KIR1.1, KIR2.1 and KIR4.1 channels. These KIR channels were expressed in HEK cells and studied in the inside-out patches under the same condition in Figure 2. None of these channels were inhibited by rosiglitazone at 30, 100 and 300 µM.
Figure 6
Figure 6
Comparison of channel inhibition between outside-out and inside-out patches. (A) In an outside-out patch, the KIR6.2/SUR2B channel was partially inhibited with external exposure to rosiglitazone (RSG) up to 300 µM. (B) With the external exposure of rosiglitazone, the relationship of KIR6.2/SUR2B channel activity with rosiglitazone concentration was shifted by ∼7-fold toward the higher concentration level, where the IC50 was 350 µM (n= 4), and 50 µM with internal exposure. (C) Similar effects were seen in the KIR6.1/SUR2B channel inhibition where the IC50 was 150 µM with external exposure (n= 15) and 10 µM with internal exposure.
Figure 7
Figure 7
The currents were recorded in the whole-cell configuration with a high concentration (145 mM) of K+ applied to either side of the plasma membrane. The membrane potential was held at 0 mV and stepped to −80 mV every 3 s as shown in the lower panel of A. The KIR6.2/SUR2B channel spontaneously opened without activator application and without ATP in the pipette solution; the channel activation was inhibited by 100 µM rosiglitazone (RSG) and further suppressed by 10 µM glibenclamide (Glib). Whole-cell currents of KIR6.1/SUR2B (B, n= 8), KIR6.2/SUR2B (C, n= 4) and KIR6.2ΔC36 channels (D, n= 5) were inhibited by 30, 100 and 100 µM rosiglitazone respectively. The current inhibition was significantly smaller than that seen in inside-out patches.
Figure 8
Figure 8
Single-channel kinetic analysis. Single-channel activity of the KIR6.2ΔC36 channel was studied in inside-out patches. (A) KIR6.2ΔC36 single-channel current at baseline. (B) The KIR6.2ΔC36 channel was inhibited by 100 µM rosiglitazone (RSG). (C) The dwell-time histogram of channel openings was described by a single-exponential with the constant τO= 2.3 ms. (D) The dwell-time histogram for channel closures contained three components of time constants: τC1= 0.5 ms, τC2= 12.7 ms, τC3= 74.6 ms. (E,F) With 100 µM rosiglitazone treatment, the dwell-time histograms of the channel openings and closures did not show marked changes (τO= 2.2 ms, τC1= 0.6 ms, and τC2= 15.9 ms) except τC3= 136.4 ms.

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References

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